Plasma high density lipoprotein (HDL)elicit their anti-atherogenic effect by transporting cholesterol from arterial macrophages to the liver for degradation, a process known as reverse cholesterol transport (RCT). Current HDL therapies are inadequate and new interventions for increasing plasma HDL levels and RCT are needed. HDL contains exchangeable apolipoproteins (apos) that are potential targets for HDL therapy. These include apo A-l,the major protein, and apos A-ll and E, which contain cysteines that are the sites of apo homo/heterodimerization. This project will fill gaps in our knowledge about HDL assembly, structure, and properties by testing new hypotheses about a] the mechanistic coordination of HDL phospholipidation and the dimerization of apos E3 and A-ll,b] the configurations of apos on HDL subfractions sorted by size and apo composition, and c] the hepatic production and phospholipidation of HDL-apos. These hypotheses will be addressed by the three specific aims: Aim 1: To use chemical kinetics to identify physico-chemical determinants of apo dimerization. This aim addresses a fundamental question about intracellular oxidative folding that is applicable to other cysteine-containing lipophilic proteins, including apo B. Aim 2: To use peptide mapping with and without chemical cross-linking and mass spectrometry to determine the apo configurations on native and reconstituted HDL. The configurations of apos on various HDL species determine HDL interactions with cell surface lipid transporters and with hepatic HDL receptors. Aim 3: To identify the itinerary of nascent HDL speciation in hepatocytes with respect to apo phospholipidation and in the case of cysteine-containing apos, the homo/heterodimerization of apos A-lland E, by steady state and pulse chase labeling methods. Fibrates, which increase apo A-llsynthesis, produce modest elevations of HDL-C, an effect that might occur through altered HDL biogenesis. Distinct mechanisms for assembly of each apo into HDL could provide distinct therapeutic targets for increasing plasma HDL-C and RCT. Obesity-linked diabetes, a serious and growing public health problem in the United States, is associated with a cluster of lipid risk factors that include low plasma HDL-C. Although niacin and the statin class of hypolipidemic drugs can increase HDL-C, their effects are modest and better HDL therapies are needed. The information derived from our research would permit a rational approach to the development of new interventions that could operate at the level of the three main components of RCT-hepatic HDL production, remodeling in the plasma compartment, and hepatic HDL-cholesterol uptake.